Platelet alpha-granules were previously known to contain at least three proteins, which are absent from plasma and are secreted upon clotting: (a) platelet factor 4 (PF4), which binds heparin; (b) another protein that can be cleaved into the so-called low-affinity platelet factor 4 (LA-PF4), which has a weaker ability to bind heparin; and (c) platelet-derived growth factor (PDGF).
The role and properties of PF4 were understood only to a limited extent because PF4 has a short half-life in serum and is not present in an uncomplexed state in any significant amounts. It was known that, upon secretion from platelets, PF4 is combined with chondroitin sulfate (although the factor is thought to have even greater affinity for heparin). The factor was also known to combine with heparan sulfate on the luminal surface of endothelial cells. Other known PF4 properties are its ability to inhibit collagenase activity and to enhance serum elastase activity, and its chemotactic activity for human leukocytes and especially fibroblasts.
Osterman et al (Biochem. Biophys. Res. Comm. 107:130-135, 1982) disclosed that a carboxy-terminal tridecapeptide was a potent chemotactic agent (inducing the directed movement of cells in response to a chemical gradient) for monocytes. The synthetic 13 amino acid residue peptide thus comprised the chemotactic active site of PF4 for monocytes.
No immunomodulatory or immunoregulating function had ever been ascribed to any peptide fragments of PF4.
In addition to antibodies, serum and ascites fluid contain a number of factors that can affect immune responses. These factors, which include products of lymphocytes, macrophages and other cells of the hemopoietic system, modulate the immune responses of an organism in a negative or a positive fashion.
Inability or diminished ability to mount immune responses is often present in mammals. It can be the direct result of a pathological condition (such as viral infection, lymphoma, and carcinoma), the treatment of such a condition (such as chemotherapy which is used to treat cancer), or the natural consequence of the aging process.
Immunosuppressed individuals are vulnerable to infection and often succumb to a secondary infection rather than the primary pathological condition from which they suffer. It is, therefore, desirable to devise methods for avoiding or overcoming immune suppression.
To study suppressed immune responses, researchers have established experimental systems in which a state of immune suppression is induced. For example, Katz, I. et al (J. Nat'l. Canc. Inst. 72:125, 1984) describe a system where mice are injected intravenously (i.v.) with gamma-irradiated syngeneic lymphoma cells (gamma-RCS) together with sheep erythrocytes (SRBC). Such mice exhibit a markedly suppressed plaque-forming cell (PFC) response. Katz, et al., supra attributed this suppression to a strong proliferation of suppressor T-cells.
The present inventors have previously found that administration of normal mouse and human serum completely reversed immuneresponse suppression in mice injected with gamma-RCS and SRBC.
They further found that T-lymphocytes bound the immunorestorative activity because both spleen cells, (a source rich in mature T-lymphocytes), and cloned cytotoxic T-lymphocytes absorbed the activity from serum.
Neither spleen cells from nude mice (T-deficient) nor thymus cells (deficient in mature T-lymphocytes) were cable of absorbing the immunorestorative activity.
The present inventors have identified platelet alphagranules as the source of the immunorestorative activity and platelet factor 4 as the molecule responsible for such activity. The activity was absent in plasma, or in serum prepared in the absence of platelets, or in serum prepared from a patient lacking platelet alpha-granules. By contrast, an activity with very similar immunorestorative properties was released by isolated human or mouse platelets, after they were stimulated with thrombin.
The present inventors have established that the immunomodulating factor was in fact PF4 (hereinafter alternatively referred to as the holoprotein) by showing the following:
1. The active substance was an alpha-granule constituent (it is absent in serum from alpha-granule-deficient patients) with affinity for heparin.
2. Other known alpha-granule constituents, LA-PF4 and PDGF, had no significant immunomodulating activity.
3. Antiserum to PF4 neutralized the immunomodulating affinity in normal human serum.
4. Isolated PF4 had as much immunomodulating activity as serum or platelet releasate when used in amounts comparable to those believed to be contained in these fluids.
This has been disclosed in the following publications:
Katz, I.R. et al J. Immunol. 134:3199-3203 1985, Katz, I.R. et al Cellular Immunol. 100:57-65 1986, Katz, I.R. et al Proc. Natl. Acad. Sci. U.S.A. 83:3491-3455 1986, all incorporated herein by reference.
In light of the above, it would be desirable to obtain fragments of the PF4 molecule capable of expressing the biologic activities of the holoprotein. Such fragments can be economically and easily obtained using conventional solid phase synthesis technique well-known in the art.